Stop Sign-Triggered Nucleic Acid Structural Transformation Enables Intracellular GlycoRNA Spatial Imaging for Evaluating of Glycosylation Mechanism.

Glycosylation is a widespread modification process in living organisms. Besides proteins and lipids, very recent research has revealed that small noncoding RNAs can also be glyco-modified, but their biosynthetic pathways and functions within cells remain unknown due to the lack of analytical strategies and intracellular spatial imaging techniques capable of detecting low-abundance targets in the presence of high levels of analogous interfering RNAs. Herein, we designed a stop sign-triggered nucleic acid structural transformation strategy to monitor spatial distribution and the potential glycosylation sites of glycoRNAs to evaluate their presumable formation mechanism. Upon the binding of the special microRNA to the artificially designed RNA code via sequence specificity, toehold-mediated strand displacement induced structural transformation of the RNA code. With special glycomodification on microRNA, it could act as a stop sign to inhibit the strand displacement in a special transition state with fluorescent RNA (FLRNA) domain to realize in situ visualization of glycoRNAs in cells. Without glycomodification, the strand displacement was continued that broke the FLRNA domain to avoid interferences of normal microRNA that generate the analysis of special glycomodification on microRNA in the presence of multitudinous normal microRNAs. Furthermore, the distribution and expression of glycoRNAs by varying regulators were revealed to provide insights into biosynthetic pathways and functions of glycoRNAs within cells. These findings suggested the potential process of N-glycan of glycoRNAs with an oligosaccharyltransferase, contributing to further exploration of glycoRNA biology.